US20230053129A1 - Compound for the treatment of the hemolytic-uremic syndrome - Google Patents

Compound for the treatment of the hemolytic-uremic syndrome Download PDF

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US20230053129A1
US20230053129A1 US17/788,688 US202017788688A US2023053129A1 US 20230053129 A1 US20230053129 A1 US 20230053129A1 US 202017788688 A US202017788688 A US 202017788688A US 2023053129 A1 US2023053129 A1 US 2023053129A1
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dab
nab815
shiga toxin
thr
mammal
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Maurizio Brigotti
Domenica Carnicelli
Elisa Porcellini
Elisabetta Galassi
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Universita di Bologna
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a compound for a use in the treatment and/or in the prevention of the hemolytic-uremic syndrome and/or in the treatment of a mammal having at least one Shiga toxin in the blood.
  • the invention further relates to the use of a compound for manufacturing a pharmaceutical preparation for the treatment and/or the prevention of the hemolytic-uremic syndrome.
  • Shiga toxins are bacterial proteins consisting of a pentamer of B subunits bound in a non-covalent manner to an A subunit, which represents the enzymatically active part (Paton e Paton 1998 Clin. Microbiol. Rev. 11, 450-479).
  • the Shiga toxins produced by bacteria such as Escherichia coli and Shigella are the crucial pathogenicity factor for the development of the hemolytic-uremic syndrome (HUS) (Tarr et al., 2005 Lancet 365, 1073-1086).
  • HUS hemolytic-uremic syndrome
  • the hemolytic-uremic syndrome is a severe consequence of these bacterial infections which affects approximately 10-15% of infected patients, especially children under the age of 3 years (Tarr et al., 2005 Lancet 365, 1073-1086).
  • HUS can manifest itself in the form of isolated cases, micro-epidemics (dozens of patients) (Tarr et al., 2005 Lancet 365, 1073-1086) or macro-epidemics (hundreds/thousands of patients), which is what happened in 2011 in Germany and in other European countries (4000 cases of infection, 800 cases of HUS, 50 deaths) (Scheutz et al., 2011 Euro. Surveill. 16).
  • HUS is characterized by hemolytic anemia, thrombocytopenia and acute renal failure, which manifest themselves approximately one week after the infection, when Shiga toxins get into the circulation. Indeed, the toxins are produced by the bacteria in the intestine and are subsequently released into the bloodstream where they interact with circulating cells (early toxemia) before determining the intoxication of the endothelia of the kidneys and of the brain, as well as of other different renal cells, triggering HUS (late toxemia) (Tarr et al., 2005 Lancet 365, 1073-1086).
  • the target cells express the glycolipid receptor globotriaosylceramide (Gb3Cer), which interacts with the pentamer of B subunits of Shiga toxins (Bauwens et al., 2011 Thromb. Haemost. 105, 515-528).
  • Gb3Cer glycolipid receptor globotriaosylceramide
  • the interaction with the circulating cells during the early toxemia plays a crucial role in the pathogenesis of the syndrome (HUS) and can take place by means of the Gb3Cer expressed on monocytes and platelets (van Setten et al., 1996 Blood 88, 174-183; Karpman et al., 2001 Blood 97, 3100-3108).
  • TLR4 Toll-like receptor 4
  • Shiga toxin 2 is the variant that is most frequently associated with the development of HUS (Friedrich et al., 2002 J Infect. Diseases 185, 74-84). Shiga toxin 2 associated with extracellular vesicles is present in the patients infected by E. coli producing Shiga toxins during the early toxemic phase (before the development of the HUS) (Brigotti et al., 2020 Thrombosis and Haemostasis 120, 107-120). Furthermore, Shiga toxin 2 can be associated with these extracellular vesicles through the A chain binding to the membrane TLR4.
  • Polymyxin B is an antibiotic active against Gram-negative bacteria (Vaara, M. 2010 Curr. Opin. Microbiol. 13, 574-581) and capable of blocking the interaction between a microbial component known as endotoxin (or lipopolysaccharide) and the TLR4 (Morrison et al., 1976. Immunochemistry 13, 813-818; Srimal et al., 1996 Biochem. J. 315, 679-686; Bannatyne et al., 1977 J. Infect. Dis. 136: 469-474).
  • endotoxin or lipopolysaccharide
  • polymyxin B proved to be capable of also impairing the Shiga toxin 1/TLR4 and Shiga toxin 2/TLR4 interaction (Carnicelli et al., 2016 J. Immunol. 196, 1177-1185).
  • polymyxin B is nephrotoxic and neurotoxic (Vaara, 2013 J. Antimicrob. Chemother. 68: 1213-1219) and effective in blocking satisfactorily the interaction between Shiga toxin 2 and the white blood cells only at high concentrations ( ⁇ g/ml).
  • the object of the invention is to provide a compound for a use in the treatment and/or in the prevention of the hemolytic-uremic syndrome and/or in the treatment of a mammal having at least one Shiga toxin in the blood, which at least partially overcomes the drawbacks of the prior art and, at the same time, is easy and economic to be implemented.
  • a further object of the invention is to provide a use of a compound for manufacturing a pharmaceutical preparation for the treatment and/or the prevention of the hemolytic-uremic syndrome, which at least partially overcomes the drawbacks of the prior art and, at the same time, is easy and economic to be implemented.
  • a compound for a use in the treatment and/or in the prevention of the hemolytic-uremic syndrome and/or in the treatment of a mammal having at least one Shiga toxin in the blood and a use of a compound for manufacturing a pharmaceutical preparation for the treatment and/or the prevention of the hemolytic-uremic syndrome according to the appended independent claims and, preferably, according to any one of the claims directly or indirectly depending on the independent claims.
  • FIG. 1 shows the effect of NAB741 (derivative of polymyxin B) on the binding of Shiga toxin 2 to the human neutrophil;
  • the ordinate shows the percentage of Shiga toxin 2 bound to the neutrophils;
  • the abscissa shows the concentration of NAB741 ( ⁇ g/ml) used;
  • FIG. 2 shows the effect of NAB7061 (derivative of polymyxin B) on the binding of Shiga toxin 2 to the human neutrophil;
  • the ordinate shows the percentage of Shiga toxin 2 bound to the neutrophils;
  • the abscissa shows the concentration of NAB7061 ( ⁇ g/ml) used;
  • FIG. 3 shows the effect of NAB815 (derivative of polymyxin B) on the binding of Shiga toxin 2 to the human neutrophil;
  • the ordinate shows the percentage of Shiga toxin 2 bound to the neutrophils;
  • the abscissa shows the concentration of NAB815 ( ⁇ g/ml) used; ***P ⁇ 0.001 (Student test) relative to the control sample with toxin and without NAB815;
  • FIG. 4 shows the percentage of Shiga toxin 2 bound to the human neutrophil as a function of the log base 10 of the concentration of NAB815; the ordinate shows the percentage of Shiga toxin 2 bound to the neutrophils relative to the value obtained in the presence of Shiga toxin 2 without NAB815; the abscissa shows the log base 10 of the concentration of NAB815 ( ⁇ g/ml) used; the IC 50 was calculated by the least squares method applying the linear regression between the percentage of Shiga toxin 2 and the logarithm of the concentration of NAB815; the Pearson coefficient (r) was used to assess the correlation between the variables;
  • FIG. 5 shows the effect of the presence of the NAB815 relative to the interaction between Raji cells and Shiga toxin 2 (Stx2); the continuous line and the dotted line indicate the extent of the protein synthesis in Raji cells treated with different concentrations of Shiga toxin 2 in the absence and in presence of NAB815, respectively; the ordinate shows the percentages of protein synthesis obtained in Raji cells with different concentrations of Shiga toxin 2 relative to the control without toxin; the abscissa shows the log base 10 of the concentrations (pM—picomolar) of Shiga toxin 2 used;
  • FIG. 8 shows the percentage of neutrophil/platelet aggregates with respect to samples treated with toxin (blood from three different human healthy donors); the ordinate shows the percentage of aggregates relative to the total aggregates obtained by adding the sole Shiga toxin 2 (Stx2a) to the sample; the different columns (from the left to the right) show the results obtained for a sample treated with Shiga toxin 2 (Stx2a), Stx2a plus polymyxin B (PMX), Stx2a plus NAB815, contaminating bacterial endotoxin (LPS) and Shiga toxin 2 treated for 30 minutes at 95° C.
  • toxin blood from three different human healthy donors
  • FIG. 9 shows the percentage of monocyte/platelets aggregates with respect to samples treated with toxin (blood from three different human healthy donors); the ordinate shows the percentage of aggregates relative to the total aggregates obtained by adding the sole Shiga toxin 2 (Stx2a) to the sample; the different columns (from the left to the right) show the results obtained for a sample treated with Shiga toxin 2 (Stx2a), Stx2a plus polymyxin B (PMX), Stx2a plus NAB815, contaminating bacterial endotoxin (LPS) and Shiga toxin 2 after treatment for 30 minutes at 95° C.
  • toxin blood from three different human healthy donors
  • the NAB815 compound (or a pharmaceutically acceptable salt thereof) for the treatment (and/or the prevention) of the hemolytic-uremic syndrome.
  • the NAB815 compound (or a pharmaceutically acceptable salt thereof) is provided for the treatment (and/or the prevention) of the hemolytic-uremic syndrome of a mammal (more in particular, of a human being).
  • the NAB815 (or a pharmaceutically acceptable salt thereof) is provided for the (for a use in the) treatment of a mammal (in particular, a human being) having at least one Shiga toxin (in particular, a Shiga toxin 2) in the blood within the circulatory system of the mammal.
  • a mammal in particular, a human being
  • the Shiga toxin in particular, the Shiga toxin 2 present in the blood within the circulatory system of the mammal (in particular, of the human being) is associated with white blood cells and/or platelets so as to form aggregates and/or extracellular vesicles.
  • a pharmaceutically acceptable salt of the NAB815 compound for the treatment (and/or) the prevention of the hemolytic-uremic syndrome.
  • the pharmaceutically acceptable salt of the NAB815 is provided for the (for a use in the) treatment of a mammal (in particular, a human being) having at least one Shiga toxin (in particular, a Shiga toxin 2) in the blood within the circulatory system of the mammal.
  • a mammal in particular, a human being
  • the Shiga toxin in particular, the Shiga toxin 2 present in the blood within the circulatory system of the mammal (in particular, of the human being) is associated with white blood cells and/or platelets so as to form aggregates and/or extracellular vesicles.
  • the NAB815 has the following formula (I):
  • L is OA
  • R1 is -Dab
  • R2 is -Thr
  • R3 is -DThr
  • R4 is -Dab
  • R5 is -Dab
  • R6 is -DPhe
  • R7 is -Leu
  • R8 is -Abu
  • R9 is -Dab
  • R10 is -Thr.
  • sequence R1-R10 represents the sequence Dab-Thr-DThr-cy[Dab-Dab-DPhe-Leu-Abu-Dab-Thr-], namely Sequence NO. 1.
  • the NAB815 is OA-Dab-Thr-DThr-cy[Dab-Dab-DPhe-Leu-Abu-Dab-Thr-], namely OA-Sequence NO. 1.
  • the NAB815 is typically associated with one or more counterions (for example, the sulphate ion) so as to form a salt (in particular, a pharmaceutically acceptable salt).
  • counterions for example, the sulphate ion
  • Dab is ⁇ , ⁇ -diamino-n-butyryl (i.e. 2,4-diaminobutyryl); Abu is 2-aminobutyryle; Thr is L-threonine; DThr is D-threonine; DPhe is D-phenylalanine; Leu is L-leucine; DSer is D-serine; OA is octanoyl; MOA is methyloctanoyl; MHA is methylheptanoyl; Ac is acyl; and cy[ . . . ] indicates a cycle consisting of the components indicated in the square brackets and in which the first and the last components are bound.
  • “pharmaceutically acceptable salt” means a salt which maintains the biological properties of the original compound.
  • methods for the preparation of these salts include the following methods: addition of inorganic acids (for example, hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid and the like) or organic acids (for example, acetic acid, oxalic acid, maleic acid, methanesulfonic acid, salicylic acid, succinic acid, citric acid and the like) to a free base of the original compound; replacement of an acid proton of the original compound with a metal cation (for example, a cation of an alkali metal or of an aluminium or the like); transfer of an acid proton of the original compound to an organic base (for example, dimethylamine, triethylamine and the like) and coordination with said organic base.
  • inorganic acids for example, hydrochloric acid, hydrobromic acid, sulphuric acid, phosphoric acid and the like
  • organic acids for example,
  • compositions are acid addition salts obtained from the use of non-toxic acids, such as hydrochloric acid, nitric acid, sulphuric acid, phosphoric acid, oxalic acid, fumaric acid, maleic acid, succinic acid, acetic acid, citric acid, ascorbic acid, maleic acid, benzoic acid, tartaric acid, carbonic acid and the like.
  • An acid typically used for the formation of the pharmaceutically acceptable salt is sulphuric acid.
  • prodrug means an agent which, in vivo, is converted into a pharmacologically active substance.
  • a prodrug can have some advantages compared to the corresponding pharmacologically active substance. For instance, it can be easier to be administered to patients and/or have a greater solubility and/or a better ability to go through cellular membranes.
  • possible prodrugs are also meant to be comprised.
  • the NAB815 can be synthesized according to what disclosed in example 1 of patent EP3045469.
  • the NAB815 (or a pharmaceutically acceptable salt thereof) has proved to have a cytotoxicity, in particular for human renal tubule cells (IC 50 334 ⁇ g/ml), approximately 20 times lower than polymyxin B (IC 50 18 ⁇ g/ml) (Vaara et al. 2017 Peptides 91, 8-12).
  • NAB815 leads to a decrease in the risk of nephrotoxicity caused by drug.
  • the structure of the NAB815 substantially differs from the one of polymyxin B (pentacationic cyclic lipodecapeptides, 3 charges in the cyclic portion, 2 in the linear portion, table 1) because of the reduction of the positive charges responsible for the toxicity from 5 to 3, 2 of them being in the cyclic portion of the molecule, unlike other tricationic cyclic lipononapeptide derivatives with 3 positive charges in the cyclic portion: NAB7061 (Vaara et al. 2008 Antimicrob. Agents Chemother. 52:3229-3236) and NAB741 (Vaara et al. 2010 Antimicrob Agents Chemother. 54: 3341-3346) (table 1).
  • Table 1 below indicates the meaning of the different parts of formula (I) for the aforesaid compounds.
  • sequence R4-R10 for polymyxin B, for NAB7061 and for NAB741 represents the sequence cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. a Sequence NO. 2. Therefore, in other words: polymyxin B is MOA/MHA-Dab-Thr-Dab-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. MOA/MHA-Dab-Thr-Dab-Sequence NO.
  • NAB7061 is OA-Thr-Abu-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], i.e. OA-Thr-Abu-Sequence NO. 2; NAB741 is Ac-Thr-DSer-cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-], e.i. Ac-Thr-DSer-Sequence NO. 2.
  • the Dabs (with the exception of the Dab of R4, i.e. the Dab determining the formation of the cyclic portion) have a respective positive charge each.
  • polymyxin B, NAB7061 and NAB741 are typically associated with one or more counterions (for example, the sulphate ion) so as to form a salt (in particular, a pharmaceutically acceptable salt).
  • counterions for example, the sulphate ion
  • the NAB815 is surprisingly more active than polymyxin B, NAB7061 and NAB741 for the treatment and/or the prevention of the hemolytic-uremic syndrome.
  • the NAB815 has proved to be surprisingly and significantly more active in inhibiting the interaction of the Shiga toxin 2 with the TLR4 in human circulating cells (human neutrophil— FIGS. 1 - 4 ); directly interact with the Shiga toxin 2; be surprisingly and significantly more active in avoiding the consequences of the interaction between the Shiga toxin 2 and the human circulating cells (blocking the formation of monocyte/platelet and neutrophil/platelet aggregates— FIGS. 6 - 9 ).
  • the NAB815 is effective in exceptionally small quantities.
  • the NAB815 can be used with a low risk of it releasing further Shiga toxins in the blood of the patient.
  • the NAB815 since it can operate in very small quantities, surprisingly manages to overcome the technical prejudice according to which antibiotics have been considered to be harmful (worsening) for patients infected by bacteria producing Shiga toxins or affected by hemolytic-uremic syndrome.
  • a use of the NAB815 compound (or a pharmaceutically acceptable salt thereof) for manufacturing a pharmaceutical preparation for the treatment (and/or the prevention) of the hemolytic-uremic syndrome Alternatively or in addition, there is provided a use of the NAB815 compound (or a pharmaceutically acceptable salt thereof) for manufacturing a pharmaceutical preparation for the treatment of a mammal (in particular, a human being) having at least one Shiga toxin (in particular, a Shiga toxin 2) in the blood within the circulatory system (of the mammal—in particular, of the human being).
  • the Shiga toxin (in particular, the Shiga toxin 2) present in the blood within the circulatory system is associated with white blood cells and/or platelets so as to form aggregates and/or extracellular vesicles in the blood within the circulatory system of the mammal (more precisely, of the human being).
  • a pharmaceutical preparation comprising a compound having general formula (I), as defined above, or a pharmaceutically acceptable salt thereof (and, in particular, a pharmaceutically acceptable excipient and/or diluent).
  • the pharmaceutical preparation comprises a pharmaceutically acceptable salt of the NAB815.
  • the pharmaceutical preparation (comprising the NAB815), according to some non-limiting embodiment, is for administration through a route chosen in the group consisting of: parenteral route, enteral route, topical route (or a combination thereof).
  • the pharmaceutical preparation further comprises one or more pharmaceutically acceptable excipients.
  • the pharmaceutical preparation can be a tablet and comprise, as pharmaceutically acceptable excipient, fructose for food use.
  • the pharmaceutical preparation (comprising the NAB815) is for subcutaneous administration, intravenous administration, intra-articular administration, intrathecal administration, intramuscular administration, intraperitoneal administration, intradermal administration (intradermal injections), transdermal administration, rectal administration, buccal administration, oromucosal administration, nasal administration, ocular administration, oral administration, inhalation and/or implant.
  • the pharmaceutical preparation (comprising the NAB815) is for parenteral administration by injection or continuous administration (according to what is known for other similar compounds).
  • injection formulations can be in the form of unitary doses, for example in bulbs or multi-dose containers containing preservatives.
  • the pharmaceutical preparation can be in the form of suspension, in watery or oily liquids, and can contain elements of the formulation as dispersion and stabilization agents.
  • the NAB815 can be in powder and be dissolved, right before use, in a liquid suited for the purpose, for example sterilized water.
  • the pharmaceutical preparation (comprising the NAB815) is administered through topical administration (on the skin) of the composition and/or through oral administration.
  • topical administration on the skin
  • oral administration the composition is in a liquid watery form (solution, syrup, drops, etc.) or in a solid form (tablets, pills, capsules, etc.).
  • the pharmaceutical preparation can be, for example, in the form of tablets or capsules prepared by means of known methods with pharmaceutically acceptable excipients as binding agents (e.g. pregelatinized corn starch, polyvinylpyrrolidone or methylcellulose), fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate), additives (e.g. magnesium stearate, talc, silica), disintegrating agents (e.g. potato starch) and/or lubricating agents (e.g. sodium lauryl sulfate).
  • binding agents e.g. pregelatinized corn starch, polyvinylpyrrolidone or methylcellulose
  • fillers e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • additives e.g. magnesium stearate, talc, silica
  • disintegrating agents e.g. potato starch
  • lubricating agents e.g. sodium lauryl sulfate
  • Liquid preparations for oral administrations can have, for example, the form of solutions, syrups or suspensions or they can be in the form of a dry product which can be dissolved in water or in another liquid before use.
  • These preparations can be prepared, in known ways, with pharmaceutically acceptable excipients as suspending agents (e.g. sorbitol, cellulose derivatives, edible hydrogenated fats), emulsifiers (e.g. lecithin or acacia), non-watery liquids (e.g. almond oil, oil esters, ethyl alcohol or fractionated vegetable oils) and/or preservatives (e.g. methyl- or propyl p-hydroxybenzoates, sorbic acid or ascorbic acid).
  • the preparations can also contain, in suitable cases, buffer salts, colouring, aromatic and/or sweetening agents.
  • Oral administration preparations can be formulated in a known manner, so as to release the active compound in a controlled manner.
  • the pharmaceutical preparation (comprising the NAB815) can be designed (in a known manner) to be administered through rectal administrations, such as suppositories or bulb syringes, for example containing known suppository excipients, such as cocoa butter or other glycerides.
  • the pharmaceutical preparation (comprising the NAB815) can be formulated (in a known manner) as a prolonged-release composition.
  • These prolonged-release compositions can be administered, for example, by means of an implant (for example a subcutaneous or intramuscular implant) or by means of an intramuscular injection.
  • the pharmaceutical preparation (comprising the NAB815) comprises suitable polymer and/or hydrophobic materials (e.g. an emulsion or an oil) and/or ion-exchange resins and/or derivatives (of the NAB815) which are relatively scarcely soluble, such as relatively scarcely soluble salts.
  • the pharmaceutical preparation can be formulated for administrations through a (known) device, for example in powder for a suitable transporter.
  • the pharmaceutical preparation only comprises NAB815 (in particular, a salt thereof) as active compound; alternatively, it comprises one or more further active ingredients, in particular an antibacterial agent.
  • the NAB815 can be formulated in a suitable preparation; suitable administration forms comprise, for example, solutions, dispersions, suspensions, powders, capsules, tablets, pills, controlled-release capsules, controlled-release tablets and controlled-release pills.
  • a method for the treatment and/or the prevention of the hemolytic-uremic syndrome in a mammal comprises administering a dose of NAB815 (or of a pharmaceutically acceptable salt thereof) to the mammal.
  • a method for the treatment of a mammal having at least one Shiga toxin 2 associated with white blood cells and/or platelets so as to form aggregates and/or extracellular vesicles in the blood within the circulatory system of the mammal (in particular, of the human being).
  • the method comprises administering a dose of NAB815 (or of a pharmaceutically acceptable salt thereof) to the mammal.
  • Examples of mammal that can be treated are: farm animals, such as cows, pigs, sheep, goats and horses; pets, such as cats and dogs; lab animals, such as guinea pigs, rabbits, mice and rats; human beings.
  • the NAB815 can be administered (to mammals—in particular, to human beings) in different ways, for example by parenteral route, topical route and/or enteral route.
  • the NAB815 can be administered through subcutaneous administration, intravenous administration, intra-articular administration, intrathecal administration, intramuscular administration, intraperitoneal administration and intradermal administration as well as through transdermal administration, rectal administration, buccal administration, oromucosal administration, nasal administration, ocular administration, through inhalation and through oral administration.
  • the dosage of the NAB815 depends on the age and conditions of the patient; therefore, the dosage should be decided case by case by the physician.
  • the dosage also depends on the administration method. Usable doses can range, for example, from 0.1 mg/Kg to 300 mg/Kg (in particular, from 0.1 mg/Kg to 100 mg/Kg; more in particular, from 0.1 mg/Kg to 30 mg/Kg), relative to the body weight, per day.
  • the NAB815 can be administered in combination with one or more suitable therapeutic agents formulated in a known and usable manner.
  • the method comprises administering a dose of NAB815 or of a pharmaceutically acceptable salt thereof to the mammal.
  • a method for the treatment of a mammal having at least one Shiga toxin 2 associated with white blood cells and/or platelets so as to form aggregates and/or extracellular vesicles in the blood within the circulatory system of the being; the method comprises administering a dose of NAB815 or of a pharmaceutically acceptable salt thereof to the human being.
  • NAB815 is effective at much smaller concentrations than polymyxin B in preventing the binding of Shiga toxin 2 to circulating cells (human neutrophils) expressing the TLR4 and in stopping the following functional consequences implied in the development of the hemolytic-uremic syndrome.
  • human neutrophils 99.7% of purity isolated in sterile conditions with low contamination of bacterial endotoxin were obtained from buffy coats of healthy donors after centrifugation on Ficoll-Paque followed by sedimentation with dextran, hypotonic lysis of the erythrocytes and positive removal of contaminating cells by means of EasySep Human Neutrophil Enrichment Kit (Stemcell Technologies, Vancouver, BC, Canada), as described above (Brigotti et al., 2013 J Immunol. 191, 4748-4758).
  • the determination of the amount of Shiga toxin 2 bound to the neutrophils was carried out through incubation with a mouse monoclonal antitoxin antibody (IgG) in the presence of human serum to avoid non-specific binding, washing with PBS and subsequent addition of an anti-murine IgG fluorescent sheep antibody (FITC).
  • IgG mouse monoclonal antitoxin antibody
  • FITC anti-murine IgG fluorescent sheep antibody
  • the fluorescence-based flow cytometry analysis allows for a detection of the fluorescence associated to neutrophils (Tazzari et al., 2004 Cytometry B Clin. Cytom. 61, 40-44).
  • the MCV parameter (mean channel value of fluorescence) of the obtained fluorescence histograms was chosen for the quantitative determination of the binding of the Shiga toxin 2 to the neutrophil.
  • NAB815 proved to be scarcely effective as inhibitors of the binding of Shiga toxin 2 to the neutrophil, as indicated by the non-significant results and by the fact that the effect is not dose-dependent ( FIGS. 1 and 2 ).
  • NAB815 proved to be a strong inhibitor of the neutrophil/Shiga toxin 2 interaction, thus determining reproducible results with a marked dose-dependent relationship even at concentrations below 1 ⁇ g/ml ( FIGS. 3 and 4 ).
  • the NAB815 is a surprisingly good inhibitor of the interaction between Shiga toxin 2 and TLR4.
  • the IC 50 concentration determining a 50% inhibition calculated by these experiments for the NAB815 (0.057 ⁇ g/ml) surprisingly is approximately 60 times lower than that obtained with polymyxin B (3.5 ⁇ g/ml) in similar conditions (Carnicelli et al. 2016 J. Immunol. 196, 1177-1185).
  • NAB815 directly interacts with the Shiga toxin 2.
  • the natural fluorescence of the Shiga toxin 2 due to the 12 tryptophan residues present in the compound.
  • the emission results were corrected for the dilution of the concentration of the toxin due to the addition of the antibiotic calculated at each point by means of spectrophotometric evaluation.
  • NAB815 there was a maximum reduction of the emission by approximately 10%, at 1:1 toxin/NAB815 stoichiometric ratio and a dissociation constant (Kd) of the complex of 0.5 ⁇ 10 ⁇ 8 M, indicating a significant affinity of the NAB815 for the Shiga toxin 2.
  • Kd dissociation constant
  • the partial reduction of the emission (10%) can be explained with the interaction of the NAB815 with the A chain of the Shiga toxin 2.
  • the B pentamer contains 10 tryptophans (2 in each B subunit); hence, the quenching of one of these tryptophans in each one of the 5 B chains, caused by the NAB815, would have determined a much more significant reduction of the emission and a non-stoichiometric toxin/NAB815 ratio.
  • the A chain only contains 2 tryptophans out of the 12 present in the holotoxin; therefore, a partial reduction of the fluorescence emission can be explained with the interaction of the NAB815 with the A chain of the Shiga toxin 2.
  • the small mass of the NAB815 (Mw 1319.44) compared to Shiga toxin 2 (Mw 68000) is in accordance with the quenching of one single tryptophan of the A chain. It should be pointed out that human cells such as the Raji cells (Burkitt lymphoma), which express the same receptor (Gb3Cer) interacting with the B chain pentamer of the Shiga toxins, as well as the in vivo target cells, are not protected by the NAB815 ( FIG. 5 ).
  • the intoxication was evaluated through the inhibition of the protein syntheses (measured in the presence of a radioactive amino acid; Arfilli et al., 2015 Toxins (Basel) 7, 4564-4576) obtained in Raji cells treated with different concentrations of Shiga toxin 2 in the presence and in the absence of NAB815 (0.3 ⁇ g/ml).
  • the NAB815 has no protective effect.
  • the NAB815 like polymyxin B, specifically inhibits TLR4/toxin interactions.
  • NAB815 blocks the functional consequences of the Shiga toxin 2/TLR4 interaction in human circulating cells.
  • NAB815 The most important effect of the NAB815 was obtained in the formation of white blood cell/platelet aggregates observed in patients during the early toxemic phase (before the development of the HUS), which are correlated with the formation of the extracellular vesicles involved in the development of the HUS.
  • the formation of these aggregates was evaluated by direct fluorescence-based flow cytometry as previously described (Carnicelli et al., 2016 J. Immunol. 196, 1177-1185). In brief, non-fractionated blood samples (1 ml) of healthy donors were incubated for 4 h at 37° C. with Shiga toxin 2 (1 nM).
  • FIGS. 6 to 9 show the effect of polymyxin B (PMX) and of NAB815 on the formation of neutrophil/platelet or monocyte/platelet aggregates in the human blood treated with Shiga toxin 2 (Stx2a).
  • FIGS. 6 and 7 show the results obtained with the blood of a representative donor; more precisely, the number of aggregates formed is expressed as percentage relative to the total number of monocytes and neutrophils, respectively.
  • FIGS. 8 and 9 show the results obtained with the blood of three different donors (data expressed as percentage of formed aggregates).
  • Sequence NO. 1 (i.e., Dab-Thr-DThr-cy[Dab-Dab-DPhe-Leu-Abu-Dab-Thr-]) corresponds to SEQ ID NO:1.
  • Sequence NO. 2 (i.e., cy[Dab-Dab-DPhe-Leu-Dab-Dab-Thr-]) corresponds to SEQ ID NO:2.
  • the sequence R1-R10 of Polymyxin B corresponds to SEQ ID NO:3.
  • sequence R2-R10 of NAB7061 corresponds to SEQ ID NO:4.
  • sequence R2-R10 of NAB741 corresponds to SEQ ID NO:5.

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